Treatment of glomerulonephritis with decorin

ABSTRACT

A method of treating of glomerulonephritis in a patient by administering decorin.

This application is a continuation of application Ser. No. 07/212,702,filed Jun. 28, 1988 now abandoned.

This invention relates to cell biology and more specifically to thecontrol of cell proliferation.

Under normal circumstances, cell proliferation is a tightly controlledprocess; fast proliferation is needed during embryonal development andtissue regeneration, whereas the proliferation must be halted in thecompleted tissue. Cell proliferation appears to be controlled primarilyby growth factors. Most of the known growth factors are stimulatory.Examples include epidermal growth factor, platelet-derived growthfactor, various interleukins and colony-stimulating factors. A fewnegative regulators of cell proliferation are also known. Transforminggrowth factor beta is a multifunctional factor that inhibits the growthof some cell types, but can also stimulate proliferation. Other growthinhibitors include various interferons and a growth inhibitory role hasalso been ascribed to heparin, heparan sulfate and their fragments.

A less well understood mechanism of growth control relates to the closeapposition of cells. Normal cells stop growing when they make contactwith one another. This phenomenon, commonly known as contact inhibitionof growth, is of obvious importance for the formation of orderly tissuestructure.

A number of important pathological conditions depend on abnormal cellproliferation. The foremost of such conditions is, of course, cancer.Other diseases with a proliferative component include rheumatoidarthritis with its overgrowth of the synovial tissue,glomerulonephritis, in which the mesangial cells proliferate, andatherosclerosis, in which the abnormally proliferating cells are smoothmuscle cells.

It is obvious from these examples that there is a great need to developnew methods for controlling cell proliferation. The present inventionaddresses this need and provides other related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to the proteglycan Decorin (also known asPG-II or PG-40). The invention provides cells transfected with andexpressing the gene coding for Decorin, and recombinant Decorin producedthereby. Spent culture media from such transfected cell cultures can beused to suppress the proliferation of either normal or abnormal cells.Moreover, purified Decorin can be used to suppress cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C show the expression of decorin in unamplified andamplified transfectants by using fluorography of SDS-7%-polyacrylamidegel electrophoresis under reducing conditions. FIG. 1A shows ³⁵ SO₄-labeled culture supernatants immunoprecipitated with rabbit antipeptideantiserum prepared against the NH₂ -terminus of human decorin. FIG. 1Bis total ³⁵ SO₄ -labeled products secreted into the culture medium. FIG.1C is total ³ H leucine labeled products secreted into culture medium.

FIG. 2 is a radiogram showing the expression of Decorin core protein inCHO cells.

FIG. 3 shows morphological changes caused by expression of Decorin inCHO cells.

FIG. 4 is a graph showing the growth of Decorin-expressing and controlCHO cells in culture.

FIG. 5 is a photomicrograph showing the effect of spent culture media onthe morphology of CHO cells.

FIG. 6 is a photomicrograph showing the effect of spent culture media onthe morphology of Harvey ras gene-transformed NIH 3T3 cells.

FIG. 7 shows the elution pattern from DEAE-Sepharose of spent culturemedia from Decorin-expressing cell line, clone 61.

FIG. 8 shows the nucleotide and deduced amino acid sequence of thedecorin set forth in FIG. 2 of Krusius and Ruoslahti, Proc. Natl. Acad.Sci. USA, 83:7683; Nucleotide and amino acid residues are numbered fromthe amino terminus of the mature core protein. Nucleotides upstream fromthe amino terminus and amino acids in the signal and propeptidesequences are indicated by negative numbers. Signal and propeptidesequences are shown underlined (thin line) and the possiblesignal-peptidase cleavage site is indicated by an arrow. Potentialglycosylation sites for N-linked glycans and glycosaminoglycans areshown by solid and open triangles, respectively. Serine at position 4 isknown to carry a glycosaminoglycan. A 48-residue homologous sequencepresent twice in the core protein is shown underlined (bold line). Thealternative 3' flanking sequence contained in clone 3C (lower line) isshown below the sequence of clone 5E (upper line) starting at base 1122where the sequences diverge. Possible polyadenylylation signal sequencesare underlined.

DETAILED DESCRIPTION OF THE INVENTION

This invention stems from work performed to explore the functions of aproteoglycan, Decorin. Proteoglycans are proteins that carry one or moreglycosaminoglycan chains. The known proteoglycans carry out a variety offunctions and are found in a variety of cellular locations. Many ofthem, however, are components of extracellular matrix, where theyparticipate in the assembly of cells to the matrix and affect theattachment of cells to the matrix.

Decorin, also known as PG-II or PG-40, is a small proteoglycan producedby fibroblasts. Its core protein has a molecular weight of about 40,000daltons. The core has been sequenced (Krusius and Ruoslahti, Proc. Natl.Acad. Sci. USA 83:7683 (1986); Day et al. Biochem. J. 248:801 (1987),both of which are incorporated herein by reference) and it is known tocarry a single glycosaminoglycan chain of a chondroitin sulfate/dermatansulfate type (Pearson, et al., J. Biol. Chem. 258:15101 (1983), which isincorporated herein by reference). The only previously known functionfor Decorin is its binding to type I and type II collagen and the effectit has on the fibril formation by this collagen (Vogel, et al., Biochem.J. 223:587 (1984)).

A molecular biological study of Decorin has now led to unexpectedobservations on its role in the control of cell proliferation, and theseobservations form the basis of this invention.

Decorin cDNA is transfected into cells, such as Chinese hamster ovary(CHO) cells, preferably those which are dihydrofolate reductase(dhfr)-negative, although other cells such as 3T3 and COS cells can alsobe used. Such transfection is accomplished by methods well-known in theart. The transfected cells are then grown in culture.

Chinese hamster ovary (CHO) cells into which human Decorin cDNA wastransfected and which express the proteoglycan from this cDNA appearmore adhesive to the substratum than the original cells. Moreover, thegrowth of the cells that expressed Decorin from the cDNA was suppressedand they grew to a lower saturation density than the various controlcells. These controls included cells transfected with a constructexpressing the core protein of Decorin and amplified to the same degreeas the Decorin expressing cells. These cells were similar to theoriginal CHO cells. The magnitude of the growth and adhesion changes wasproportional to the amount of Decorin produced.

Moreover, changes in the adhesion and the saturation density could bereproduced with the spent culture media of the cells expressing therecombinant Decorin and with the Decorin isolated and purified from suchculture media. These findings indicate that Decorin plays a previouslyunsuspected role in the control of cell proliferation, and that it canbe used to modulate cell proliferation. The effect seen withoncogene-transformed 3T3 cells suggests that this invention may beuseful in the treatment of proliferative diseases.

As used herein "Decorin" refers to a proteoglycan having the structuralcharacteristics attributed to it in Krusius and Ruoslahti, supra, andwhich suppresses cell proliferation as determined by the method ofExample III. Human fibroblast Decorin has substantially the amino acidsequence presented in Krusius and Ruoslahti supra, FIG. 2, which isincorporated herein by reference. "Decorin" refers both to the nativecomposition and to modifications thereof which retain the functionalcharacteristics.

The recombinant Decorin of the invention has a structure correspondingsubstantially to that of the native proteoglycan. It is understoodhowever that limited modifications may be made however withoutdestroying the Decorin activity.

EXAMPLE I Expression of Decorin and Decorin Core Protein

The 1.8 kb full-length Decorin cDNA described in Krusius and Ruoslahti,Proc. Natl. Acad. Sci. USA 83:7683 (1986), which is incorporated hereinby reference, was used for the construction of Decorin expressionvectors. For the expression of Decorin core protein, a mutagenized cDNAin which the fourth codon, TCT coding for serine, was changed to ACTcoding for threonine was engineered by site-directed mutagenesisaccording to the method of Kunkel, Proc. Natl. Acad. Sci USA 82:488(1985), which is incorporated herein by reference. The mammalianexpression vectors pSV2-Decorin and pSV2-Decorin/CP (core protein) wereconstructed by ligating the Decorin cDNA or the mutagenized Decorin cDNAinto 3.4 kb HindIII-Bam HI fragment of pSV2 (Mulligan and Berg, Science209:1423 (1980) which is incorporated herein by reference),respectively. Dihydrofolate reductase (dhfr)-negative CHO cells(CHO-DG44) were cotransfected with pSV2-Decorin or pSV2-Decorin/CP andpSV2dhfr by the calcium phosphate coprecipitation method. Thetransfected cells were cultured in nucleoside-minus alpha-modifiedminimal essential medium (α-MEM, GIBCO, Long Island) supplemented with9% dialyzed fetal calf serum, 2 mM glutamine, 100 units/ml penicillinand 100 μg/ml streptomycin. Colonies arising from transfected cells werepicked using cloning cylinders, expanded and checked for the expressionof Decorin by immunoprecipitation from ³⁵ SO₄ -labeled culturesupernatants. Clones expressing a substantial amount of Decorin werethen subjected to gene amplification by stepwise increasingconcentration of methotrexate (MTX, Kaufman and Sharp, J. Mol. Biol.159:601 (1982) which is incorporated herein by reference) up to 0.64 μM.All the amplified cell lines were cloned either by limiting dilution orby picking single MTX resistant colonies. Stock cultures of theseestablished cell lines were kept in MTX-containing medium. Before use inexperiments, cells were subcultured in MTX-minus medium from stockcultures and passed at least once in this medium to eliminate thepossible MTX effects. Controls were transfected only with pSV2dhfr andtreated exactly as experimental cells thereafter. Metabolic labeling ofthe cells with ³⁵ SO₄ or ³ H-leucine and immunoprecipitation wasperformed as described Brennan et al., J. Biol. Chem 259:13742 (1984),which is incorporated herein by reference.

FIG. 1 shows the expression of Decorin in unamplified and amplifiedtransfectants, by using fluorography of SDS-7%-polyacrylamide gelelectrophoresis under reducing conditions. (A) ³⁵ SO₄ -labeled culturesupernatants immunoprecipitated with rabbit antipeptide antiserumprepared against the NH₂ -terminus of human Decorin (Krusius andRuoslahti, supra.). (B) Total ³⁵ SO₄ -labeled products secreted intoculture medium. (C) Total ³ H-leucine labeled products secreted intoculture medium. Lane 1: control transfectant A, an unamplified clonetransfected with pSV2dhfr; lane 2: control transfectant C, a cloneamplified to 0.64 lambda M MTX resistance from control transfectant A;lane 3: clone 1, an unamplified primary transfectant expressing 0.2pg/cell/day of Decorin; lane 4: clone 31, a clone amplified to 0.32lambda M MTX resistance and expressing 4 pg/cell/day of Decorin; lane 5:clone 61, a clone amplified to 0.64 lambda M MTX resistance andexpressing 25 pg/cell/day of Decorin.

FIG. 2 shows expression of Decorin core protein in CHO cells. Lanes 1and 2: ³ H-leucine-labeled culture supernatants were immunoprecipitatedas described in FIG. 1. Lanes 3 and 4: Total ³ H-leucine-labeledproducts secreted into culture medium. Lanes 1 and 3: CHO cellstransfected with pSV2-Decorin/CP. Lanes 2 and 4: Control CHO cellstransfected with pSV2dhfr.

EXAMPLE II Quantitation of Cell Spreading and Saturation Density

The cell lines of Example I were plated in 24 well plates in MTX-minusculture medium at a density of 3×10⁵ cells per well. After 24 hours,medium was replaced (0.3 ml per well) and cells were incubated another24 hours. Concentration of Decorin in these culture supernatants wasdetermined by competitive ELISA (Engvall, Meth. Enzymol. 70:419 (1980)which is incorporated herein by reference). Briefly, a mixture ofculture supernatant and rabbit antipeptide antibody against Decorin wasincubated in the wells of microtiter plates coated with Decorin purifiedfrom human fetal membranes (Brennan et al., supra.). The amount ofantibody bound to the wells was determined byalkaline-phosphatase-conjugated goat anti-rabbit IgG as a secondantibody. Various concentrations of purified Decorin were used togenerate a standard curve. The cells were counted by hemocytometer atthe end of the 24 hour incubation.

As shown in Table I, cells transfected with the Decorin gene exhibited alarger area of spreading than did control cells. Where Decorinexpression was amplified, area of spreading increased with increasingexpression.

Also shown in Table I are the saturation densities of theDecorin-expressing and control cells. In order to determine thesaturation densities, cells (1.2×10⁵) were plated in a 60 mm culturedish in MTX-minus culture medium. After 6 hours, cells were fixed with3% paraformaldehyde and stained with toluidine blue. Quantitativeevaluation of spreading was performed by measuring the surface area ofthe cells with a surface integration program of an image analyzer(Olympus). Nonspread cells were excluded from the measurement. The meanand standard deviation of values from 50 cells are shown.

The CHO-DG44 cells transfected with pSV-decorin have been deposited withthe American Type Culture Collection (ATCC) 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. on Jan. 22, 1990 under ATCC Accession No.CRL 10332. This deposit was pursuant to the requirements of the BudapestTreaty on the International Recognition of Deposit of Microorganisms forthe Purposes of Patent Procedure.

                                      TABLE I                                     __________________________________________________________________________    PRODUCTION OF DECORIN AND SPREADING OF TRANSFECTANTS                                            Decorin                                                                  MTX  Prod.        Saturation                                                  Resistance                                                                         (μg per 10.sup.6                                                                  Spread Area                                                                         Density                                        Clone                                                                              Transfection                                                                          (μM)                                                                            cells/day)                                                                           (μM.sup.2 /cell)                                                                 (× 10.sup.-5)                            __________________________________________________________________________    control                                                                            pSV2dhfr                                                                              0    0      2725 ± 627                                                                       10.8 ± 1.2                                  line A                                                                        control                                                                            pSV2dhfr                                                                              0.32 0      2585 ± 693                                                                       10.4 ± 2.5                                  line B                                                                        control                                                                            pSV2dhfr                                                                              0.64 0      2659 ± 586                                                                       10.6 ± 1.8                                  line C                                                                        clone 1                                                                            pSV2-decorin +                                                                        0    0.2    3368 ± 842                                                                       9.9 ± 1.6                                        pSV2dhfr                                                                 clone 31                                                                           pSV2-deconrin +                                                                       0.32 4      4759 ± 898                                                                       7.3 ± 0.2                                        pSV2dhfr                                                                 clone 33                                                                           pSV2-decorin +                                                                        0.32 11     5554 ± 1002                                                                      5.2 ± 0.2                                        pSV2dhfr                                                                 clond 66                                                                           pSV2-decorin +                                                                        0.64 14     5482 ± 1382                                                                      4.9 ± 0.3                                        pSV2dhfr                                                                 clone 61                                                                           pSV2-decorin +                                                                        0.64 25     6472 ± 1147                                                                      4.4 ± 0.4                                        pSV2dhfr                                                                 __________________________________________________________________________

EXAMPLE III Analysis of the Effect of Spent Culture Media

The effect of spent culture media on the morphology of CHO cells andHarvey ras gene-transformed NIH 3T3 cells was determined by plating CHOcells in 35 mm dishes at a density of 2×10⁵ cells/dish in two-day spentmedia from clone 61 containing approximately 20 μg/ml of Decorin and insimilar media from control cell line C containing no Decorin andcultured, the cell lines being those described in Example I.

FIG. 5 shows the morphology of the CHO cells after this treatment andFIG. 6 shows the morphology of the treated oncogen-transformed 3T3cells. As can be seen, the spent culture medium from theDecorin-expressing cell line, clone 31, induced a morphology similar tothat observed in the Decorin-expressing cells themselves. Theoncogene-transformed 3T3 cells treated in this manner assume amorphology closely similar to that of normal cells. This morphology isoften referred to as "contact inhibited morphology" and it is consideredto be indicative of normal growth control. In accordance with thisphenomenon, fewer cells were seen in these cultures compared to thecontrol-media treated cultures. These results indicate that the culturemedia from the cell lines expressing Decorin reproduces themorphological and growth inhibiting effects seen in the recombinantDecorin-expressing cells themselves.

EXAMPLE IV Purification of Decorin from Spent Culture Media

Clone 61 cells were grown to 90% confluence in 8 175 cm² culture flasksin nucleoside minus α-MEM supplemented with 9% dialyzed fetal calfserum, 2 mM glutamine, 100 units/ml penicillin and 100 μg/mlstreptomycin. At 90% confluence culture media was changed to 25 ml perflask of nucleoside-free α-MEM supplemented with 6% dialyzed fetal calfserum which had been passed through a DEAE Sepharose Fast Flow column(Pharmacia) equilibrated with 0.25M NaCl in 0.05M phosphate buffer, pH7.4. Cells were cultured for 3 days, spent media was collected andimmediately made to 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/mlpepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA.

Four hundred milliliters of the spent media were first passed throughgelatin-Sepharose to remove fibronectin and materials which would bindto Sepharose. The flow-through fraction was then mixed withDEAE-Sepharose preequilibriated in 50 mM Tris/HCl, pH 7.4, plus 0.2MNaCl and batch absorbed overnight at 4° C. with gentle mixing. Theslurry was poured into a 1.6×24 cm column, washed extensively with 50 mMTris/HCl, pH 7.4, containing 0.2M NaCl and eluted with 0.2M-0.8M lineargradient of NaCl in 50 mM Tris/HCl, pH 7.4. Decorin concentration wasdetermined by competitive ELISA as described above.

FIG. 7 shows the elution pattern in DEAE-Sepharose Fast Flow. As can beseen, Decorin separates from the bulk of the protein present in themedia and can be recovered in substantially pure form from the fractionsshowing the highest immune reactivity.

Although the invention has been described with reference to thepresently-preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A method of treating glomerulonephritis in a patient, said method comprising administering decorin to said patient.
 2. The method of claim 1, wherein said decorin comprises the mature core protein set forth as amino acids 1-329 of FIG.
 8. 3. The method of claim 1, wherein said decorin is produced recombinantly. 